Immunizing composition and method for inducing an immune response against the β-secretase cleavage site of amyloid precursor protein

ABSTRACT

The present invention is directed to an immunizing composition containing an antigenic product such as a multiple antigen peptide system (MAPS) or a filamentous bacteriophage displaying an AβPP epitope spanning the β-secretase cleavage site of AβPP and a method for inducing an immune response against the β-secretase cleavage site of AβPP using this immunizing composition. The present invention is also directed to antibodies against the β-secretase cleavage site of AβPP and their use in a method for inhibiting the formation of amyloid β.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to an immunizing composition and methodfor inducing an immune response against β-secretase cleavage site ofamyloid precursor protein. The present invention further relates toantibodies raised or generated against the β-secretase cleavage site ofamyloid precursor protein and the use thereof in passive immunization.

2. Description of the Related Art

Amyloid Precursor Protein and β-Secretase:

The extracellular deposition of short amyloid peptides in the brains ofpatients is thought to be a central event in the pathogenesis ofAlzheimer's disease. Evidence that amyloid may play an important role inthe early pathogenesis of AD comes primarily from studies of individualsaffected by the familial form of AD (FAD) or by Down's syndrome. Thegeneration of amyloid β peptide (Aβ) occurs via a regulated cascade ofcleavage in its precursor protein, AβPP (amyloid precursor protein). Atleast three enzymes are responsible for AβPP proteolysis and have beententatively named α, β, and γ secretase. The recent identification ofseveral of these secretases is a major leap in understanding how thesesecretases regulate amyloid peptide formation. One of the maintherapeutic goals is the inhibition of secretases that produce Aβ fromthe large precursor protein. The theoretical specificity andtractability of protease targets suggest that it should be possible togenerate secretase-specific protease inhibitors that penetrate the bloodbrain barrier. Many studies using new knowledge of the ability of theβ-secretase enzyme (BACE) to identify inhibitors by screening orrational design approaches are already underway (U.S. Pat. Nos.5,744,346; 5,942,400; 6,221,645 B1; 6,313,268 B1; and published PCTapplications WO 00/47618, WO 98/21589, and WO 96/40885). At this point,there is no evidence of additional functions of Aβ, so there are noserious concerns about reduction of this metabolite. Both β- andγ-secretases are present in many different cells in the body and it isreasonable to assume that they have substrates in addition to AβPP.Consequently, complete inhibition of one of these enzymes might resultin toxicity problems, particularly under the chronic treatmentconditions that would presumably be required. At the mRNA level, BACE isexpressed widely in the human brain. Expression is also high in thepancreas, although enzymatic activity in this tissue is low. Apart fromAβPP cleavage, it is not known if BACE possesses other activity and soit is too early to predict what toxicity β-secretase inhibitors mayhave.

Proteolytic processing of the amyloid precursor protein (AβPP) generatesamyloid β (Aβ) peptide which is thought to be causal for the pathologyand subsequent cognitive decline in Alzheimer's disease. To initiate Aβformation, β-secretase cleaves AβPP at the N-terminus of Aβ to releaseAPPsβ, an approximately 100-kD soluble N-terminal fragment, and C99, a12-kD C-terminal fragment which remains membrane bound. The exact siteof β-secretase cleavage has been determined (FIG. 1). Amyloid plaque Aβstarts at Aspl and this cleavage site is therefore of major interest.Cleavage by β-secretase at the amino terminus of the Aβ peptidesequence, between residues 671 and 672 of AβPP, leads to the generationand extracellular release of β-cleaved soluble AβPP, and a correspondingcell-associated carboxy-terminal fragment.

One of the familial AD families was shown to have a mutation in AβPPthat coincided with the predicted cleavage site of β-secretase. Thisdouble mutation, first identified in a Swedish pedigree, was also foundto mechanistically result in overproduction of Aβ peptide relative towild sequence when it was transfected into cells, suggesting that it wasa better substrate for the β-secretase enzyme. This prediction hasrecently been borne out to be true. A Met to Leu substitution at the Plposition of APP, found in the “Swedish” familial AD mutation whichcauses early-onset AD, dramatically enhances β-secretase cleavage, butmany other substitutions (for example, Met to Val) decrease β-secretasecleavage. These findings demonstrated the presence of a β-secretaseactivity responsible for a cleavage event that liberated the N terminusof Aβ peptide and showed the process was secretory rather thanlysosomal, the favored hypothesis at the time.

Blood Brain Barrier:

The blood-brain barrier (BBB) (Johansson, 1992; Ermisch, 1992;Schlosshauer, 1993) is formed by a monolayer of tightly connectedmicrovascular endothelial cells with anionic charges. This layerseparates two fluid-containing compartments: the blood plasma (BP) andextracellular fluid (ECF) of the brain parenchyma, and is surrounded byastroglial cells of the brain. One of the main functions of the BBB isto regulate the transfer of components between the BP and the ECF. TheBBB limits free passage of most agent molecules from the blood to thebrain cells.

In general, large molecules of high polarity, such as peptides,proteins, (e.g., enzymes, growth factors and their conjugates,oligonucleotides, genetic vectors and others) do not cross the BBB.Therefore poor agent delivery to the CNS limits the applicability ofsuch macromolecules for the treatment of neurodegenerative disorders andneurological diseases.

Several delivery approaches of therapeutic agents to the braincircumvent the BBB. Such approaches utilize intrathecal injections,surgical implants (Ommaya, 1984 and U.S. Pat. No. 5,222,982) andinterstitial infusion (Bobo et al., 1994). These strategies deliver anagent to the CNS by direct administration into the cerebrospinal fluid(CSF) or into the brain parenchyma (ECF).

Drug delivery to the central nervous system through the cerebrospinalfluid is achieved by means of a subdurally implantable device namedafter its inventor, the “Ommaya reservoir”. The reservoir is used mostlyfor localized post-operative delivery of chemotherapeutic agents incancers. The drug is injected into the device and subsequently releasedinto the cerebrospinal fluid surrounding the brain. It can be directedtoward specific areas of exposed brain tissue which then adsorb thedrug. This adsorption is limited since the drug does not travel freely.A modified device developed by Ayub Ommaya, whereby the reservoir isimplanted in the abdominal cavity and the injected drug is transportedby cerebrospinal fluid (taken from and returned to the spine) all theway to the ventricular space of the brain, is used for agentadministration.

Diffusion of macromolecules to various areas of the brain byconvection-enhanced delivery is another method of administrationcircumventing the BBB. This method involves: a) creating a pressuregradient during interstitial infusion into white matter to generateincreased flow through the brain interstitium (convection supplementingsimple diffusion); b) maintaining the pressure gradient over a lengthyperiod of time (24 hours to 48 hours) to allow radial penetration of themigrating compounds (such as: neurotrophic factors, antibodies, growthfactors, genetic vectors, enzymes, etc.) into the gray matter; and c)increasing drug concentrations by orders of magnitude over systemiclevels. Through their direct infusion into the brain parenchyma, thesite-specific biomolecular complexes of U.S. Pat. No. 6,005,004 deliverthe agent to neuronal or glial cells, as needed, and be retained bythese cells. Moreover, the site-specific complexes containing neuronaltargeting or internalization moieties are capable of penetrating theneuronal membrane and internalizing the agent.

Another strategy to improve agent delivery to the CNS is by increasingthe agent absorption (adsorption and transport) through the BBB andtheir uptake by the cells (Broadwell, 1989; Pardridge et al., 1990;Banks et al., 1992; and Pardridge, edited by Vranic et al., 1991. Thepassage of agents through the BBB to the brain can be enhanced byimproving either the permeability of the agent itself or by altering thecharacteristics of the BBB. Thus, the passage of the agent can befacilitated by increasing its lipid solubility through chemicalmodification, and/or by its coupling to a cationic carrier, or still byits covalent coupling to a peptide vector capable of transporting theagent through the BBB. Peptide transport vectors are also known as BBBpermeabilizer compounds (U.S. Pat. No. 5,268,164).

Phage Display:

Combinatorial phage display peptide libraries provide an effective meansto study protein:protein interactions. This technology relies on theproduction of very large collections of random peptides associated withtheir corresponding genetic blueprints (Scott et al, 1990; Dower, 1992;Lane et al, 1993; Cortese et al, 1994; Cortese et al, 1995; Cortese etal, 1996). Presentation of the random peptides is often accomplished byconstructing chimeric proteins expressed on the outer surface offilamentous bacteriophages such as M13, fd and f1. This presentationmakes the repertoires amenable to binding assays and specializedscreening schemes (referred to as biopanning (Parmley et al, 1988))leading to the affinity isolation and identification of peptides withdesired binding properties. In this way peptides that bind to receptors(Koivunen et al, 1995; Wrighton et al, 1996; Sparks et al, 1994;Rasqualini et al, 1996), enzymes (Matthews et al, 1993; Schmitz et al,1996) or antibodies (Scott et al, 1990; Cwirla et al, 1990; Felici etal, 1991; Luzzago et al, 1993; Hoess et al, 1993; Bonnycastle et al,1996) have been efficiently selected.

Filamentous bacteriophages are nonlytic, male specific bacteriophagesthat infect Escherichia coli cells carrying an F-episome (for review,see Model et al, 1988). Filamentous phage particles appear as thintubular structures 900 nm long and 10 nm thick containing a circularsingle stranded DNA genome (the +strand). The life cycle of the phageentails binding of the phage to the F-pilus of the bacterium followed byentry of the single stranded DNA genome into the host. The circularsingle stranded DNA is recognized by the host replication machinery andthe synthesis of the complementary second DNA strand is initiated at thephage ori(−) structure. The double stranded DNA replicating form is thetemplate for the synthesis of single-stranded DNA circular phagegenomes, initiating at the ori(+) structure. These are ultimatelypackaged into virions and the phage particles are extruded from thebacterium without causing lysis or apparent damage to the host.

Peptide display systems have exploited two structural proteins of thephage; pIII protein and pVIII protein. The pIII protein exists in 5copies per phage and is found exclusively at one tip of the virion(Goldsmith et al, 1977). The N-terminal domain of the pIII protein formsa knob-like structure that is required for the infectivity process (Grayet al, 1981). It enables the adsorption of the phage to the tip of theF-pilus and subsequently the penetration and translocation of the singlestranded phage DNA into the bacterial host cell (Holliger et al, 1997).The pIII protein can tolerate extensive modifications and thus has beenused to express peptides at its N-terminus. The foreign peptides havebeen up to 65 amino acid residues long (Bluthner et al, 1996; Kay et al,1993) and in some instances even as large as full-length proteins(McCafferty et al, 1990; McCafferty et al, 1992) without markedlyaffecting pIII function.

The cylindrical protein envelope surrounding the single stranded phageDNA is composed of 2700 copies of the major coat protein, pVIII, anα-helical subunit which consists of 50 amino acid residues. The pVIIIproteins themselves are arranged in a helical pattern, with the α-helixof the protein oriented at a shallow angle to the long axis of thevirion (Marvin et al, 1994). The primary structure of this proteincontains three separate domains: (1) the N-terminal part, enriched withacidic amino acids and exposed to the outside environment; (2) a centralhydrophobic domain responsible for: (i) subunit:subunit interactions inthe phage particle and (ii) transmembrane functions in the host cell;and (3) the third domain containing basic amino acids, clustered at theC-terminus, which is buried in the interior of the phage and isassociated with the phage-DNA. pVIII is synthesized as a precoat proteincontaining a 23 amino acid leader-peptide, which is cleaved upontranslocation across the inner membrane of the bacterium to yield themature 50-residue transmembrane protein (Sugimoto et al, 1977). Use ofpVIII as a display scaffold is hindered by the fact that it can toleratethe addition of peptides no longer than 6 residues at its N-terminus(Greenwood et al, 1991; Iannolo et al, 1995). Larger inserts interferewith phage assembly. Introduction of larger peptides, however, ispossible in systems where mosaic phages are produced by in vivo mixingthe recombinant, peptide-containing, pVIII proteins with wild type pVIII(Felici et al, 1991; Greenwood et al, 1991; Willis et al, 1993). Thisenables the incorporation of the chimeric pVIII proteins at low density(tens to hundreds of copies per particle) on the phage surfaceinterspersed with wild type coat proteins during the assembly of phageparticles. Two systems have been used that enable the generation ofmosaic phages; the “type 8+8” and “type 88” systems as designated bySmith (Smith, 1993).

The “type 8+8” system is based on having the two pVIII genes situatedseparately in two different genetic units (Felici et al, 1991; Greenwoodet al, 1991; Willis et al, 1993). The recombinant pVIII gene is locatedon a phagemid, a plasmid that contains, in addition to its own origin ofreplication, the phage origins of replication and packaging signal. Thewild type pVIII protein is supplied by superinfecting phagemid-harboringbacteria with a helper phage. In addition, the helper phage provides thephage replication and assembly machinery that package both the phagemidand the helper genomes into virions. Therefore, two types of particlesare secreted by such bacteria, helper and phagemid, both of whichincorporate a mixture of recombinant and wild type pVIII proteins.

The “type 88” system benefits by containing the two pVIII genes in oneand the same infectious phage genome. Thus, this obviates the need for ahelper phage and superinfection. Furthermore, only one type of mosaicphage is produced.

The phage genome encodes 10 proteins (pI through pX) all of which areessential for production of infectious progeny (Felici et al, 1991). Thegenes for the proteins are organized in two tightly packedtranscriptional units separated by two non-coding regions (Van Wezenbeeket al, 1980). One non-coding region, called the “intergenic region”(defined as situated between the pIV and pII genes) contains the (+) andthe (−) origins of DNA replication and the packaging signal of thephage, enabling the initiation of capsid formation. Parts of thisintergenic region are dispensable (Kim et al, 1981; Dotto et al, 1984).Moreover, this region has been found to be able to tolerate theinsertion of foreign DNAs at several sites (Messing, 1983; Moses et al,1980; Zacher et al, 1980). The second non-coding region of the phage islocated between the pVIII and pIII genes, and has also been used toincorporate foreign recombinant genes as was illustrated by Pluckthun(Krebber et al, 1995).

Immunization with Phage Display:

Small synthetic peptides, consisting of epitopes, are generally poorantigens requiring the chemical synthesis of a peptide and need to becoupled to a large carrier, but even then they may induce a low affinityimmune response. An immunization procedure for raising anti-AβPantibodies, using as antigen the filamentous phages displaying only EFRHpeptide, was developed in the laboratory of the present inventor(Frenkel et al., 2000 and 2001). Filamentous bacteriophages have beenused extensively in recent years for the ‘display’ on their surface oflarge repertoires of peptides generated by cloning randomoligonucleotides at the 5′ end of the genes coding for the phage coatprotein (Scott and Smith, 1990; Scott, 1992). As recently reported,filamentous bacteriophages are excellent vehicles for the expression andpresentation of foreign peptides in a variety of biologicals (Greenwoodet al., 1993; Medynski, 1994). Administration of filamentous phagesinduces a strong immunological response to the phage effects systems(Willis et al., 1993; Meola et al., 1995). Phage coat proteins pIII andpVIII discussed above are proteins that have been often used for phagedisplay. The recombinant filamentous phage approach for obtainingspecific peptide antigens has a major advantage over chemical synthesis,as the products obtained are the result of the biological fidelity oftranslational machinery and are not subject to the 70-94% purity levelscommon in the solid-phase synthesis of peptides. The phage presents aneasily renewable source of antigen, as additional material can beobtained by growth of bacterial cultures.

Immunization with the EFRH (SEQ ID NO:2) epitope displaying phage may,in a short period of time, raise the high concentration of high affinity(IgG) antibodies able to prevent the formation of β-amyloid and tominimize further toxic effects. The level of antibody in the sera wasfound to be related to the number of peptide copies per phage (Frenkelet al., 2000b).

The antibodies resulting from EFRH (SEQ ID NO:2) phage immunization aresimilar regarding their immunological properties to antibodies raised bydirect injection with whole amyloid β (Table 1). These antibodiesrecognize the full length Aβ-peptide (1-40) and exhibit anti-aggregatingproperties as antibodies raised against whole Aβ peptide and/or amyloidβ (Frenkel et al., 2000b, 2001). The high immunogenicity of filamentousphages enables the raising of antibodies against self-antigens.Immunization of guinea pigs with EFRH (SEQ ID NO:2) phage as an antigen,in which the Aβ peptide sequence is identical to that in humans,resulted in the production of self-antibodies (Frenkel et al., 2001).

TABLE 1 Competitive inhibition by various peptides within Aβ of serumantibody raised against f88-EFRH compared to amyloid anti-aggregatingantibody*. MICE anti-aggregating PEPTIDE RESIDUES SERUM antibody*. FRH(residues 4-6 of Aβ) ~10⁻³ M 3 × 10⁻³ M EFRH (residues 3-6 of Aβ; SEQ IDNO: 2) 6.0 × 10⁻⁶ M 3 × 10⁻⁶ M DAEFRH (residues 1-6 of Aβ; residues 1-6of SEQ ID NO: 3) 3.0 × 10⁻⁶ M 8 × 10⁻⁷ M DAEFRHD (residues 1-7 of Aβ;residues 1-7 of SEQ ID NO: 3) 5.0 × 10⁻⁶ M 9 × 10⁻⁷ M DAEFRHDSG(residues 1-9 of Aβ; SEQ ID NO: 3) 5.0 × 10⁻⁶ M 1 × 10⁻⁶ M Aβ(1-40) 3.0× 10⁻⁶ M 8 × 10⁻⁷ M WVLD (SEQ ID NO: 4) Nd** Nd** *Frenkel et. al. 1998**IC₅₀ value of less than 10⁻² M which cannot be detected by ELISAassay.

The above data demonstrated that a recombinant bacteriophage displayinga self-epitope can be used as a vaccine to induce autoantibodies fordisease treatment. Filamentous phages are normally grown using alaboratory strain of E. coli, and although the naturally occurringstrain may be different, it is reasonable to assume that delivery ofphage into the gut will result in infection of the natural intestinalflora. The laboratory of the present inventor has found that UVinactivated phages are as immunogenic as their infective counterparts.There is evidence of long lasting filamentous phages in the guts of theimmunized animals that may explain the long lasting immune responsefound in pIII immunized mice (Zuercher et al., 2000).

Due to the high antigenicity of the phage, administration can be givenby the intranasal route, which is the easiest way for immunizationwithout any use of adjuvant. As olfactory changes are proposed to play arole in Alzheimer's disease (Murphy, 1999) mucosal immunization is aneffective induction of specific Aβ IgA antibodies for preventing localpathologic effect of the disease.

The efficacy of phage-EFRH antigen in raising anti-aggregating β-amyloidantibodies (Solomon and Frenkel, 2000) versus whole β-amyloid showsthat:

a. the high immunogenicity of the phage enables production of high titerof IgG antibodies in a short period of weeks without need of adjuvantadministration;

b. self-expression of the antigen led to long-lasting immunization;

c. the key role of the EFRH epitope in β-amyloid formation and its highimmunogenicity led to anti-aggregating antibodies which recognize wholeβ-amyloid peptide, substituting the use of β-amyloid fibrils.

Antibody Engineering:

Antibody engineering methods were applied to minimize the size of mAbs(135-900 kDa) while maintaining their biological activity (Winter etal., 1994). These technologies and the application of the PCR technologyto create large antibody gene repertoires make antibody phage display aversatile tool for isolation and characterization of single chain Fv(scFv) antibodies (Hoogenboom et al., 1998). The scFvs can be displayedon the surface of the phage for further manipulation or may be releasedas a soluble scFv (˜25 kd) fragment.

The laboratory of the present inventor engineered an scFv which exhibitsanti-aggregating properties similar to the parental IgM molecule(Frenkel et al., 2000a). For scFv construction, the antibody genes fromthe anti-AβP IgM 508 hybridoma were cloned. The secreted antibody showedspecific activity toward the AβP molecule in preventing its toxiceffects on cultured PC 12 cells. Site-directed single-chain Fvantibodies are the first step towards targeting therapeutic antibodiesinto the brain via intracellular or extracellular approaches.

Citation of any document herein is not intended as an admission thatsuch document is pertinent prior art, or considered material to thepatentability of any claim of the present application. Any statement asto content or a date of any document is based on the informationavailable to applicant at the time of filing and does not constitute anadmission as to the correctness of such a statement.

SUMMARY OF THE INVENTION

The present invention provides an immunizing composition containing animmunizing effective amount of an antigenic product which induces animmune response against the β-secretase cleavage site of amyloidprecursor protein (AβPP).

The present invention also provides a method for inducing an immuneresponse against the β-secretase cleavage site of AβPP which involvesadministering the immunizing composition according to the presentinvention to a subject/patient in need thereof.

Further provided by the present invention is a molecule comprising theantigen binding portion of an antibody against the β-secretase cleavagesite of AβPP. This molecule according to the present invention can beused in a method for blocking β-secretase cleavage of AβPP.

A preferred embodiment of the molecule according to the presentinvention is a single chain antibody, which when displayed on thesurface of a filamentous bacteriophage display vehicle can be used in amethod for inhibiting the formation of amyloid β according to thepresent invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the amino acid sequence (SEQ ID NO:1) surrounding theβ-secretase cleavage site on AβPP, where the cleavage is between Met(M)and Asp(D), designated residues 0 and 1 based on the cleavage site andwhere residue 0 (P₁ position) is normally Met but is found to be Leu inthe “Swedish” familial AD mutation.

FIGS. 2A-2C show schematic representations of embodiments of multipleantigenic peptide (MAP) on octa-branched homo Wang resin according tothe present invention. The arrow represents the cleavage site and theISEVKMDA (residues 1 to 8 of SEQ ID NO:1, where residue 6 is Met; FIG.2A), ISEVKLDA (residues 1 to 8 of SEQ ID NO:1, where residue 6 is Leu;FIG. 2B), VKMDAEFRH (SEQ ID NO:5; FIG. 2C) antigenic peptide sequence.

FIG. 3 is a graph showing the immune response after immunization withMAP-ISEVKLDA (residues 1-8 of SEQ ID NO:1, where residue 6 is Leu).

FIG. 4 is a graph showing the inhibition of total amyloid beta peptide(Aβ) secretion to growth media after 48 hrs. as measured by ELISA.

FIG. 5 is a graph showing the inhibition of intracellular accumulationof Aβ 1-42 peptide after 5 days incubation as measured by ELISA.

FIG. 6 is a confocal microscopy image showing co-localization in theperinuclear region of anti-β secretase cleavage site antibodiesaccording to the present invention and BACE antibodies raised againstthe β-secretase enzyme itself.

FIGS. 7A and 7B are images of permeabilized (FIG. 7A) and control (FIG.7B) cells immunostained with anti-β secretase cleavage site on APPantibody and a secondary antibody.

FIG. 8 is a graph showing reduction of plaque number in transgenic miceimmunized with the antigen, compared with untreated mice.

DETAILED DESCRIPTION OF THE INVENTION

β-secretase cleavage generates the free N-terminus of Aβ and istherefore considered the first critical step in amyloid formation. Toavoid the possible problems of inhibiting the enzyme per se, which couldlead to “unknown” effects, the present inventor developed a novelapproach to block β-secretase cleavage of AβPP by generating anti-AβPPantibodies capable of blocking the cleavage site of β-secretase on AβPPto inhibit the in vivo formation of Aβ and thus inhibit or prevent thedevelopment of Alzheimer's disease.

The present invention is directed to a vaccine, which is also referredherein as an immunizing composition, containing an immunizing effectiveamount of an antigenic product that induces an immune response againstthe β-secretase cleavage site of AβPP, and to a method of using thisimmunizing composition for inducing an immune response against theβ-secretase cleavage site of AβPP. This method for inducing an immuneresponse against the β-secretase cleavage site of AβPP involvesadministering the immunizing composition according to the presentinvention to a subject/patient in need thereof.

The present invention is further directed to a method for passiveimmunization by administering a viral display vehicle exposing on itssurface at least an antigen-binding (immunological) portion of anantibody which can bind to an AβPP epitope spanning the β-secretasecleavage site of AβPP to inhibit the formation of Aβ by blockingβ-secretase cleavage of AβPP. This passive immunity may be ofexceptionally long duration if the display vehicle employed is capableof replicating within the recipient/patient.

For purposes of this specification and the accompanying claims, theterms “patient”, “subject” and “recipient” are used interchangeably.They include humans and other mammals which are the object of eitherprophylactic, experimental, or therapeutic treatment. Also, the terms“amyloid β peptide” and “β amyloid peptide” are synonymous with“β-amyloid peptide”, “βAP”, “βA”, and “Aβ”. All of these terms refer toa plaque forming peptide derived from amyloid precursor protein (AβPP).

As used herein, the term “treating” includes substantially inhibiting,slowing or reversing the progression of a disease, substantiallyameliorating clinical symptoms of a disease or substantially preventingthe appearance of clinical symptoms of a disease.

The term “immune response” or its equivalent “immunological response”refers to the development of a beneficial humoral (antibody mediated)and/or a cellular (mediated by antigen-specific T cells or theirsecretion products) response directed against an AβPP epitope spanningthe β-secretase cleavage site of AβPP in a recipient patient. Such aresponse can be an active response induced by administration ofimmunogen or a passive response induced by administration of antibody. Acellular immune response is elicited by the presentation of polypeptideepitopes in association with Class I or Class II MHC molecules, toactivate antigen-specific CD4⁺ T helper cells and/or CD8⁺ cytotoxic Tcells. The response may also involve activation of monocytes,macrophages, NK cells, basophils, dendritic cells, astrocytes, microgliacells, eosinophils or other components of innate immunity.

As used herein “active immunity” refers to any immunity conferred upon asubject by administration of an antigen.

As used herein “passive immunity” refers to any immunity conferred upona subject without administration of an antigen. “Passive immunity”therefore includes, but is not limited to, administration of areplicating display vehicle which includes anantigen-binding/immunological portion of an antibody presented on itssurface to a recipient. Although replication of such a vehicle isactive, the immune response is passive from the standpoint of therecipient.

For purposes of this specification and the accompanying claims, theterms “epitope” and “antigenic determinant” are used interchangeably torefer to a site on an antigen to which B and/or T cells respond. B-cellepitopes can be formed both from contiguous amino acids or noncontiguousamino acids juxtaposed by tertiary folding of a protein. Epitopes formedfrom contiguous amino acids are typically retained on exposure todenaturing solvents whereas epitopes formed by tertiary folding aretypically lost on treatment with denaturing solvents. An epitopetypically includes at least 3, and more usually, at least 5 or 8-10amino acids in a unique spatial conformation. Methods of determiningspatial conformation of epitopes include, for example, x-raycrystallography and 2-dimensional nuclear magnetic resonance. See, e.g.,Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66,Glenn E. Morris, Ed. (1996). Antibodies that recognize the same epitopecan be identified in a simple immunoassay showing the ability of oneantibody to block the binding of another antibody to a target antigen.T-cells recognize continuous epitopes of about nine amino acids for CD8cells or about 13-15 amino acids for CD4 cells. T cells that recognizethe epitope can be identified by in vitro assays that measureantigen-dependent proliferation, as determined by ³H-thymidineincorporation by primed T cells in response to an epitope (Burke et al.,1994), by antigen-dependent killing (cytotoxic T lymphocyte assay,Tigges et al.) or by cytokine secretion.

Preferred embodiments of the antigenic product used in the immunizingcomposition according to the present invention to induce an immuneresponse against the β-secretase cleavage site of AβPP include (1) anantigen structurally based on multiple peptide antigen system (MAPs), adendritic polymer system, in which antigenic peptides representing theβ-secretase cleavage sites are covalently bound to the branches thatradiate from a core molecule, and (2) a viral display vehicle displayingan AβPP epitope spanning the β-secretase cleavage site of AβPP on itssurface.

The antigenic product used in a preferred embodiment of the presentinvention which is structurally based on a dendritic polymer ischaracterized by a higher concentration of functional groups per unit ofmolecular volume than for ordinary polymers. Generally, dendriticpolymers are based upon two or more identical branches originating froma core molecule having at least two functional groups. Such polymershave been described by Denkewalter et al. in U.S. Pat. No. 4,289,872 andby Tomalia et al. in several U.S. patents, including U.S. Pat. Nos.4,599,400 and 4,507,466. Other polymers of the class have been describedby Erickson in U.S. Pat. No. 4,515,920. The polymers are often referredto as dendritic polymers because their structure may be symbolized as atree with a core trunk and several branches. Unlike a tree, however, thebranches in dendritic polymers are all substantially identical. Thisdendrite system has been termed the multiple antigen peptide system(MAPS), which is the commonly used name for a combinationantigen/antigen carrier that is composed of two or more, usuallyidentical, antigenic molecules covalently attached to a dendritic corewhich is composed of principal units which are at least bifunctional.Each bifunctional unit in a branch provides a base for added growth. Thedendritic core of a multiple antigen peptide system can be composed oflysine molecules and confers a high immunogenicity to the whole antigen.For example, a lysine is attached via peptide bonds through each of itsamino groups to two additional lysines. This second generation moleculehas four free amino groups each of which can be covalently linked to anadditional lysine to form a third generation molecule with eight freeamino groups. A peptide may be attached to each of these free groups toform an octavalent multiple peptide antigen (MAP; FIG. 2). The processcan be repeated to form fourth or even higher generations of molecules.With each generation, the number of free amino groups increasesgeometrically and can be represented by 2^(n), where n is the number ofthe generation. Alternatively, the second generation molecule havingfour free amino groups can be used to form a tetravalent MAP, i.e., aMAP having four peptides covalently linked to the core. Many othermolecules, including e.g., aspartic acid and glutamic acid, both ofwhich have two carboxyl groups and one amino group to producepolyaspartic or polyglutamic acids with 2^(n) free carboxyl groups, canbe used to form the dendritic core of a multiple antigen peptide system.

As will be apparent from the discussion hereinafter, some of the carrieror core molecules used to form the product of the present invention areof a molecular weight such that they might not usually be regarded aspolymers. However, since their basic structure is similar to dendriticpolymers, it is convenient to describe them as such. Therefore the term“dendritic polymer” will be sometimes used herein to define the productof the invention. The term includes carrier molecules which aresufficiently large to be regarded as polymers as well as those which maycontain as few as three monomers.

The necessary chemistry for performing the synthesis of dendriticpolymers is known and available. With amino acids, the chemistry forblocking functional groups which should not react and then removing theblocking groups when it is desired that the functional groups shouldreact has been described in detail in numerous patents and articles inthe technical literature. The dendritic polymers and the entire MAP canbe produced on a resin as in the Merrifield synthesis and then removedfrom the polymer. Tomalia utilized ammonia or ethylenediamine as thecore molecule. In this procedure, the core molecule is reacted with anacrylate ester by Michael addition and the ester groups removed byhydrolysis. The resulting first generation molecules contain three freecarboxyl groups in the case of ammonia and four free carboxyl groupswhen ethylenediamine is employed. Tomalia extends the dendritic polymerwith ethylenediamine followed by another acrylic ester monomer, andrepeats the sequence until the desired molecular weight is attained. Itwill, however, be readily apparent to one skilled in the art, that eachbranch of the dendritic polymer can be lengthened by any of a number ofselected procedures. For example, each branch can be extended bymultiple reactions with lysine molecules.

Erickson utilized the classic Merrifield technique in which apolypeptide of substantially any desired molecular weight is grown froma solid resin support. As the technique is utilized for the preparationof dendritic polymers, the linking molecule which joins the polymer tothe resin support is trifunctional. One of the functional groups isinvolved in the linkage to the resin, the other two functional groupsserve as the starting point for the growth of the polymer. The polymeris removed from the resin when the desired molecular weight has beenobtained. One standard cleavage procedure is treatment with liquidhydrogen fluoride at 0° C. for one hour. Another, and more satisfactoryprocedure, is to utilize a complex of hydrogen fluoride anddimethylsulfide (HF:DMF) as described by Tam et al (1983). Thisprocedure greatly minimizes side reactions and loss of peptide.

Denkewalter, in one example of his process, utilizes lysine as the coremolecule. The amino groups of the core molecule are blocked byconversion to urethane groups. The carboxyl group is blocked by reactionwith benzhydrylamine. Hydrolysis of the urethane groups generates abenzhydrylamide of lysine with two free amino groups which serve as thestarting points for the growth of the dendritic polymer. This briefoutline of three of the available procedures for producing dendriticpolymers should be adequate to teach those skilled in the art the basicprinciples of the current technology. They will also teach the skilledartisan the salient features of the polymers, one of the most importantof which is that the polymers provide a large number of availablefunctional groups in a small molecular volume. The result is that a highconcentration of antigens in a small volume can be achieved by joiningthe antigen to those available functional groups. Moreover, theresulting molecular product contains a high proportion of antigen on arelatively small carrier, i.e., the ratio of antigen to carrier is quitehigh. This is in contrast to conventional products used as a basis forvaccines. These conventional products often are composed of a smallamount of antigen on a large amount of carrier.

Other important features of the dendritic polymer as an antigen carrierare that the exact structure is known; there are no contaminants whichmay be themselves antigenic, produce tissue irritation or otherundesirable reactions; the exact concentration of the antigen is known;the antigen is symmetrically distributed on the carrier; and the carriercan be utilized as a base for more than one antigen so that multivalentvaccines can be produced. The principal advantage of MAPS as the basisfor vaccines is that unlike other systems using natural carriers such askeyhole limpet hemocyanin, tetanus toxoid and bovine serum albumin, thedendritic polymers of MAPS as carriers are fully defined chemicalentities on which the antigens are dispersed in known concentrations.Additionally, the antigen comprises a large part of the molecule, not arelatively small and undefined proportion of the molecule, as in thecase of natural carriers.

When the MAPS is to be employed to produce a vaccine, also referred toherein as an immunizing composition, it is preferred that the coremolecule be a naturally occurring amino acid such as lysine so that itcan be dealt with by the body following the usual metabolic pathways.However, as will be explained more fully hereinafter, amino acids whichare not naturally occurring, even those which are not α-amino acids canbe employed. The acids, or any other asymmetric molecules used inbuilding the core molecule can be in either the D or L form.

Although the dendritic polymers have been principally describedhereinabove as polyamide polymers, it will be readily apparent that thecarriers of this invention are not limited to dendritic polyamides. Anyof a wide variety of molecules having at least two available functionalgroups can serve as core molecules. Propylene glycol, for example, canserve as the basis for a polyester dendritic polymer. Succinic acid withselected glycols or amines can serve as a core molecule to generatepolyesters or polyamides. Diisocyanates can be used to generatepolyurethanes. The important point is that the core molecule has atleast two available functional groups from which identical branches canbe generated by sequential scaffolding type reactions with additionalmolecules also having at least two available functional or anchoringgroups on each branch. In the most simple case in which the coremolecule has two available functional groups and each succeedinggeneration has two available functional groups, the number of anchoringsites to which antigen molecules can be anchored is expressed by 2^(n),where n is the number of the generation.

For a more complete discussion of the chemistry of dendritic polymers,attention is directed to Tomalia et al. (1985), Aharoni et al. (1982),and the following U.S. Pat. Nos. 4,289,872; 4,558,120; 4,376,861;4,568,737; 4,507,466; 4,587,329; 4,515,920; 4,599,400; 4,517,122; and4,600,535.

The antigenic product used in the immunizing composition of the presentinvention, in a presently preferred embodiment, provides a multipleantigen peptide system comprising a dendritic polymer base with aplurality of anchoring sites covalently bound to antigenic moleculeswhich may be the same or different. The polymers comprise a central coremolecule having at least two functional groups to which molecularbranches having terminal functional groups are covalently bound. Theterminal functional groups on the branches are covalently bonded toantigenic molecules, principally described herein as peptide antigens.

The selected antigen may be separately synthesized or otherwise obtainedand joined to the carrier. Alternatively, the antigen may be synthesizedon the carrier. For instance, if the antigen is an oligopeptide orrelatively low molecular weight polypeptide, and the availablefunctional groups on the polymer are amino groups or carboxyl groups,the antigen can be synthesized by extending each branch of the polymerutilizing known peptide synthesis techniques.

FIGS. 2A-2C shows the structures of three embodiments of MAP dendriticpolymer on a resin which may be employed in the practice of the presentinvention. As can be seen, they are third generation dendriticpolylysine products. It may be obtained commercially, for example, as anocta-branched or tetra-branched Wang resin with a MAP core from a numberof suppliers, i.e., Advanced ChemTech, Inc. Louisville, Ky., or it maybe produced by conventional solid phase techniques by generating thepolymer on a Pam or a Pop resin. See Mitchell et al, (1978) and Tam etal, (1980). The polymer is then cleaved from the resin using, preferablyHF:DMS. The dendritic polylysine, was built from an alanine linkeroriginally joined to the resin. Other linkers such as glycine can beemployed. Of course, the linker can be omitted, or a plurality of linkermolecules can be utilized.

Peptide antigens having either residues 1-8 of SEQ ID NO:1 where residue6 is Met (FIG. 2A), residues 1-8 of SEQ ID NO:1 where residue 6 is Leu(FIG. 2B), or SEQ ID NO:5 (FIG. 2C) joined directly to each of theavailable functional groups on each terminal lysine moiety are shown inFIGS. 2A-2C. In the case when the antigen is a relatively short peptide,e.g., 6 to 14 residues, it may be useful to extend the polylysine by alinker such as a simple tri- or tetrapeptide of glycine, alanine orbeta-alanine. However, for antigenic peptides with more than 14residues, the linker is normally unnecessary.

Preferably the peptide antigens attached to each of the availablefunctional groups on the terminal moiety to form an octavalent MAP areas follows:

(MAP)—ISEVKMDA (residues 1-8 of SEQ ID NO:1 where residue 6 is Met)contains an epitope spanning the β-secretase cleavage site of AβPP innormal people; and

(MAP)—ISEVKLDA (residues 1-8 of SEQ ID NO:1 where reside 6 is Leu)contains an epitope spanning the β-secretase cleavage site of AβPP inthe Swedish mutation of AD.

The peptide antigens will be synthesized (growing from the C-terminus tothe N-terminus) on, e.g., an octa-branched Wang Resin,resin-β-Ala-Lys-2Lys-4Lys-4 Fmoc, which is a MAP core resin. Theocta-branched Wang resin can be obtained from the supplier, AdvancedChemTech, Inc., Louisville, Ky. (www.peptide.com) and has a cleavablepart consisting of beta-alanine to which seven lysines, branched like atree, are attached. The branches terminate at four lysines with two Emocgroups each for a total of eight Fmoc groups. The synthesis of MAP canbe performed according to the supplier's instructions or according toany number of peptide synthesis protocols, such as disclosed in U.S.Pat. No. 5,229,490 and Tam et al. (1989).

A preferred embodiment of the present invention has been described forconvenience, principally as applied to products built on lysine as thecore molecule. In fact, lysine and lysine-like molecules such asornithine, nor-lysine and amino alanine are preferred molecules forbuilding the product of this invention because they are relatively easyto obtain, they are easy to work with, and they afford good yields. Suchcore molecules can be represented by the general formula:

wherein x, y and z are integers from 0 to 10, preferably 0 to 4 providedthat at least one of them is 1 and the amino groups cannot be attachedto the same carbon atom. In the most preferred molecules, the total ofx, y and z is from 2 to 6 and the amino groups are separated by at leasttwo methylene groups.

Other preferred core molecules include ethylene diamine and likemolecules with longer chains such as propylene diamine and butylenediamine. Such molecules may be represented by the general formula:H₂N—CH₂—(CH₂)_(n)—CH₂—NH₂wherein n is an integer from 0 to 10, preferably 0 to 3. Of courseammonia can also be employed as a core molecule.

The development of synthetic vaccines against a large number of diseaseshas been greatly accelerated because of the recognition that a vaccineneed not be based on a native protein, but may be based on a lowmolecular weight segment of the native protein. These segments, normallycalled immunogenic determinants or epitopes are capable of stimulatingthe production of antibodies which will protect against, e.g., infectionby an infectious vector of the native protein antigen. The immunogenicdeterminants are often low molecular weight peptides which can beconveniently synthesized. If they cannot be synthesized, they may beseparated in pure form from the native protein itself.

Hereinafter, these antigenic immunostimulants will be referred to asantigenic peptides.

A principal embodiment used in the present invention may be broadlydefined as an antigenic product comprising a dendritic core molecule orpolymer to which a plurality of antigens such as antigenic peptidescontaining epitopes of the β-secretase cleavage site on AβPP arecovalently bonded to the available functional groups. The antigens orepitopes may be different, although preferably the antigens or epitopesare the same.

More specifically, a principal embodiment used in the present inventionmay be defined as an antigenic product or a carrier system comprising adendritic polymer base which is a central core molecule having at leasttwo available functional groups to which branches of selected lengthsare joined. Each branch of the molecule terminates with at least oneavailable anchoring functional group, a plurality of which areconvalently bonded to antigenic molecules.

The antigenic peptide that is covalently joined to the availableterminal functional groups on the dendritic polymer contains at leastone copy of an epitope spanning the β-secretase cleavage site of AβPP.When more than one copy of an epitope, such as two or three copies, ispresent on the antigenic peptide, a spacer of 2-8 amino acid residues,preferably 2-4 residues, separates the multiple copies of the epitope.

In a preferred embodiment used in the present invention, the epitope isISEVKMDA or ISEVKLDA (residues 1-8 of SEQ ID NO:1). For small antigenicpeptides, such as those having 6-12 residues, an octa-branched dendriticpolymer (eight terminal functional groups) such as the octa-branched MAPWang resin, is preferred. However, for larger peptides, in the range ofabout 20 amino acid residues or larger, a tetra-branched dendriticpolymer, such as the tetra-branched MAP Wang resin is preferred.

An advantage of the dendritic polymer is that it can serve as a carrierfor two or more different antigens, if desired. For instance,(MAP)-VKMDAEFRH (SEQ ID NO:5) represents a combination of the twodifferent key epitopes of AβPP, the β-secretase Met-Asp cleavage siteand the EFRH aggregating site of Aβ. One embodiment of the antigenicproduct used in the present invention is based on the use of a dendriticpolylysine or other structurally similar molecule employing differentamino blocking groups, one of which is stable to acid hydrolysis, theother of which is stable to alkaline hydrolysis. This makes it possibleto protect either of the amino groups of lysine by the orthogonalprotection method.

Fluorenylmethyloxycarbonyl (Fmoc) is a base labile protecting group andis completely stable to acidic deprotection. The t-butoxycarbonylblocking group (Boc) is stable under basic conditions but not stableunder mildly acidic conditions such as 50% trifluoroacetic acid. Bychoosing Boc-lys (Boc)-OH, Boc-lys (Fmoc)-OH, Fmoc-lys (Boc)-OH orFmoc-lys (Fmoc)-OH, it is possible to place one set of antigens on thealpha amino group of lysine and another on the omega amino group. Thoseskilled in the art of peptide synthesis can readily devise methods ofachieving the same types of products using diverse blocking groups andother dendritic polymers.

A few general observations applicable to the synthesis of MAPS will beof assistance to those skilled in the art. These are:

1. The syntheses generally require a long coupling time (2-4 hours).

2. Dimethyl formamide is generally a more suitable solvent thanmethylene dichloride.

3. The peptide resin should not be dried at any stage of the synthesissince resolvation is extremely difficult.

4. Coupling should be closely monitored for completion of the couplingby the quantitative ninhydrin method.

5. The MAPS is best cleaved from the resin by the improved aciddeprotection method with either HF or TFMSA (Tam, et al., 1983 and 1986)in dimethyl sulfide to avoid strong acid catalyzed side reactions.

6. MAPS tend to strongly aggregate after cleavage from the resinsupport. Purification is best effected by extensive dialysis under basicand strongly denaturing conditions in a dialysis medium which is 8M inurea and mercaptoethanol to remove undesirable aromatic additives of thecleavage reactions such as p-cresol and thiocresol. Furtherpurification, if desired, can be effected using high performancegel-permeation or ion exchange chromatography. In most cases the MAPScould be used directly without further purification.

It will be apparent to those skilled in the art that many variations ofthe structures shown and discussed herein are possible. All suchvariations are specifically included within the scope of this invention.For example, see U.S. Pat. No. 5,229,490, the entire content of which isincorporated herein by reference.

The antigenic product used in the present invention may include alipophilic membrane anchoring moiety that confers adjuvant propertiesamong its advantages. A lipophilic membrane-anchoring moiety at thecarboxyl terminus of MAP enables further non-covalent amplification by aliposome or micellar form. Accordingly, the immunizing composition ofthe present invention, which contains the antigenic product, may furtherbe prepared with a variety of vehicles, including encapsulation withinliposomes, for greater efficiency of delivery and concomitantly reduceddosage. The preparation of liposomes is well known in the art.

Tripalmitoyl-S-glyceryl cysteine (P3C) and palmitoyl lysine (PL) arenon-limiting examples of suitable lipophilic moieties for the antigenicproduct used in the present invention. P3C, which is a lipoamino acidfrom Escherichia coli, is a B cell mitogen that has proved particularlysuccessful as a non-toxic adjuvant. See U.S. Pat. No. 5,580,563 andDeFoort et al., (1992), the entire contents of which are incorporatedherein by reference.

Because the MAPs used in this invention as an antigenic product providesa high concentration of antigen in a small molecular volume, in manyinstances the vaccine/immunizing composition of the invention may beemployed, without adjuvants. However, if an adjuvant is employed, it maybe selected from any of those normally employed to stimulate theimmunogenic systems of mammals.

As used herein the term “adjuvant” refers to a compound that, whenadministered in conjunction with an antigen, augments the immuneresponse to the antigen, but when administered alone does not generatean immune response to the antigen. Adjuvants can augment an immuneresponse by several mechanisms including lymphocyte recruitment,stimulation of B and/or T cells, and stimulation of macrophages.

The viral display vehicle as an antigenic product used in the immunizingcomposition according to the present can be a double stranded DNA virus,a single stranded DNA virus, an RNA virus (positive or negative strand).Preferably, the display vehicle is a filamentous bacteriophage such asfd, f88, f1, and M13. Due to its linear structure, filamentous phage hashigh permeability to different kinds of membranes (Scott et al., 1990)and following the olfactory tract, it reaches the hippocampus area viathe limbic system to target affected sites. The treatment of filamentousphage with chloroform changes the linear structure to a circular one,which prevents delivery of phage to the brain.

While the fd filamentous phage is a particularly preferred phagesequence for use in the present invention, it should be understood thatall filamentous phages are very similar and have the same geneorganization (Model et al, 1988). Thus, the principles of the presentinvention can be applied to any of the filamentous phages, such as M13,f1 and others.

Preferably, the display vehicle is capable of propagation in therecipient. Thus, for example, a bacteriophage display vehicle can bepropagated in bacterial flora, such as Escherichia coli residing in therecipient's body. Alternatively, the display vehicle can be an in vivonon-propagateable particle. Although concerns about the potentialinfection of the natural intestinal flora (Delmastro et al., 1997;Willis et al., 1993; and Poul et al., 1999) have been expressed, UVinactivation of phage showed (Delmastro et al., 1997) that they are asimmunogenic as their infective counterparts. Use of inactivated phagemay preclude incorporation of phage encoded transgenes into the nucleusfor subsequent expression in host cells (Larocca et al., 1998), animportant practical consideration. Therefore, according to alternatepreferred embodiments, the display vehicles employed in the presentinvention may be either replicating or non-replicating.

Phage or virus display involves the expression of cDNA clones as fusionproteins with phage or virus coat proteins. If the cDNAs selected forexpression encode antigens, the phage or virus may then be employed asan antigen presenting vehicle, which can optionally replicate within arecipient.

Antigens displayed by a phage or virus may be used directly forvaccination, without antigen purification. In this case, the bulk of thecoat proteins serve to stimulate a general immune response because theyare “non-self” with respect to the vaccinated subject. The antigen-coatprotein fusion elicits a specific antibody against epitopes in thedisplayed cDNA gene product.

According to a preferred embodiment of the antigenic product used in theimmunizing composition according to present invention, the displayvehicle is selected such that less than 30 days following anintroduction of a triple dose of 10¹⁰ units thereof to the recipient, atiter of the antibodies in the recipient is above 1:50,000, as isdetermined by ELISA.

The vaccines/immunizing composition of the invention may be defined ascomprising a pharmaceutically acceptable carrier, excipient, adjuvant,or auxiliary agent, together with an amount of antigenic product of theinvention which is sufficient to produce an immunological response. Aneffective amount may be very small. It will, as is known, vary with theantigen. With the MAPS antigenic product of this invention, because ofthe high concentration of antigen in a low molecular volume, it will belower than with ordinary vaccines employing the same antigens. Thequantity which constitutes an effective amount may vary depending onwhether the vaccine is intended as a first treatment or as a boostertreatment.

It may be convenient to provide the products of this invention aslyophilized or freeze dried powders ready to be reconstituted with apharmaceutically acceptable carrier just prior to use.

In prophylactic applications, the immunizing composition of the presentinvention is administered to a subject/patient susceptible to, orotherwise at risk of Alzheimer's disease, in an amount sufficient to theonset of the disease, including biochemical, histologic and/orbehavioral symptoms of the disease, its complications and intermediatepathological phenotypes presenting during development of the disease.Individuals who have a known genetic risk of Alzheimer's disease includethose having relatives who have experienced this disease and those whoserisk is determined by analysis of genetic or biochemical markers.Genetic markers of risk towards Alzheimer's disease include mutations inthe APP gene, particularly mutations at positions 717 and positions 670and 671, referred to as the Hardy and Swedish mutations, respectively.In therapeutic applications, the immunizing composition of the inventionis administered to a patient suspected of, or already suffering fromAlzheimer's disease in an amount sufficient to at least partially arrestthe symptoms of Alzheimer's disease (biochemical, histologic and/orbehavioral), including its complications and intermediate pathologicalphenotypes in development of Alzheimer's disease. An amount adequate toblock β-secretase cleavage of AβPP and inhibit formation of Aβ isdefined as an effective dosage. In the method for inducing an immuneresponse against the β-secretase cleavage site of AβPP, the immunizingcomposition of the present invention is usually administered in severaldoses until a sufficient immune response has been achieved. Typically,the immune response is monitored and repeated doses are given if theimmune response starts to wane.

Effective dosages of the immunizing composition of the present inventionfor inducing an immune response against the β-secretase cleavage site ofAβPP vary depending upon many different factors, including means ofadministration, target site, physiological state of the patient, whetherthe patient is human or an animal, other medications administered, andwhether treatment is prophylactic or therapeutic. Usually, the patientis a human, but nonhuman mammals including transgenic mammals can alsobe treated. Treatment dosages need to be titrated to optimize safety andefficacy. The amount of immunogen depends on whether adjuvant is alsoadministered, with higher dosages more likely to be required in theabsence of adjuvant. The 1-500 μg per patient and more usually from5-500 μg per injection is used for human administration. Occasionally, ahigher dosage of 1-2 mg per injection is used. Typically about 10, 20,50 or 100 μg is used for each human injection. The mass of immunogenalso depends on the mass ratio of immunogenic epitope within theimmunogen to the mass of immunogen as a whole. The timing of injectionscan vary significantly from once a day, to once a year, to once adecade. On any given day that a dose of immunogen is given, the dosageis greater than 1 μg/patient and usually greater than 10 μg/patient ifadjuvant is also administered, and may be greater than 10-100 μg/patientin the absence of adjuvant. A typical regimen consists of animmunization followed by booster injections at time intervals, such as 6week intervals. Another regimen consists of an immunization followed bybooster injections 1, 2 and 12 months later. Another regimen entails aninjection every two months for life. Alternatively, booster injectionscan be on an irregular basis as indicated by monitoring of immuneresponse.

The immunizing composition of the present invention forinducing/eliciting an immune response can be administered by parenteral,topical, intravenous, oral, subcutaneous, intraarterial, intracranial,intraperitoneal, intranasal or intramuscular means for prophylacticand/or therapeutic treatment. The most typical route of administrationof an immunogenic agent is subcutaneous although other routes can beequally effective. The next most common route is intramuscularinjection. This type of injection is mostly typically performed in thearm or leg muscles.

The immunizing composition of the invention can sometimes beadministered in combination with an adjuvant. A variety of adjuvants canbe used in combination with the antigenic product of the invention toelicit an immune response. Preferred adjuvants augment the intrinsicresponse to an immunogen without causing conformational changes in theimmunogen that affect the qualitative form of the response. Preferredadjuvants include aluminum hydroxide and aluminum phosphate, 3De-O-acylated monophosphoryl lipid A (MPL™) (see GB 2220211, RIBIImmunoChem Research Inc., Hamilton, Mont., now part of Corixa).Stimulon™ QS-21 is a triterpene glycoside or saponin isolated from thebark of the Quillaja Saponaria Molina tree found in South America (seeKensil et al., 1995); U.S. Pat. No. 5,057,540 (AquilaBioPharmaceuticals, Framingham, Mass.). Other adjuvants are oil in wateremulsions (such as squalene or peanut oil), optionally in combinationwith immune stimulants, such as monophosphoryl lipid A (see Stoute etal., 1997). Another adjuvant is CpG (WO 98/40100). Alternatively, theantigenic product can be coupled to an adjuvant. However, such couplingshould not substantially change the conformation of the epitope so as toaffect the nature of the immune response thereto. Adjuvants can beadministered as a component of an immunizing composition with theantigenic product of the invention administered separately, before,concurrently with, or after administration of the adjuvant.

A preferred class of adjuvants is aluminum salts (alum), such asaluminum hydroxide, aluminum phosphate, aluminum sulfate. Such adjuvantscan be used with or without other specific immunostimulating agents suchas MPL or 3-DMP, QS-21, polymeric or monomeric amino acids such aspolyglutamic acid or polylysine. Another class of adjuvants isoil-in-water emulsion formulations. Such adjuvants can be used with orwithout other specific immunostimulating agents such as muramyl peptides(e.g., N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutarninyl-L-alanine-2-(1′-2′dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(MTP-PE),N-acetylglucsaminyl-N-acetylmuramyl-L-Al-D-isoglu-L-Ala-dipalmitoxypropylamide (DTP-DPP) theramide™), or other bacterial cell wallcomponents. Oil-in-water emulsions include (a) MF59 (WO 90/14837),containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionallycontaining various amounts of MTP-PE) formulated into submicronparticles using a microfluidizer such as Model 110Y microfluidizer(Microfluidics, Newton Mass.), (b) SAF, containing 10% Squalene, 0.4%Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP, eithermicrofluidized into a submicron emulsion or vortexed to generate alarger particle size emulsion, and (c) Ribi™ adjuvant system (RAS) (RibiImmunoChem, Hamilton, Mont.) containing 2% squalene, 0.2% Tween 80, andone or more bacterial cell wall components from the group consisting of5 monophosphoryllipid A (MPL), tiehalose dimycolate (TDM), and cell wallskeleton (CWS), preferably MPL+CWS (Detox™). Another class of preferredadjuvants is saponin adjuvants, such as Stimulon™ (QS-21, Aquila,Framingham, Mass.) or particles generated therefrom such as ISCOMs(immunostimulating complexes) and ISCOMATRIX. Other adjuvants includeComplete Freunds Adjuvant (CFA), Incomplete Freunds Adjuvant (IFA) andcytokines, such as interleukins (IL-1, IL-2, and IL-12), macrophagecolony stimulating factor (M-CSF), tumor necrosis factor (TNF).

An adjuvant can be administered with an immunogen as a singlecomposition, or can be administered before, concurrent with or afteradministration of the immunogen. Immunogen and adjuvant can be packagedand supplied in the same vial or can be packaged in separate vials andmixed before use. Immunogen and adjuvant are typically packaged with alabel indicating the intended application. If immunogen and adjuvant arepackaged separately, the packaging typically includes instructions formixing before use. The choice of an adjuvant and/or carrier depends onthe stability of the immunogenic formulation containing the adjuvant,the route of administration, the dosing schedule, the efficacy of theadjuvant for the species being vaccinated, and, in humans, apharmaceutically acceptable adjuvant is one that has been approved or isapprovable for human administration by pertinent regulatory bodies. Forexample, Complete Freund's adjuvant is not suitable for humanadministration. Alum, MPL and QS-21 are preferred. Optionally, two ormore different adjuvants can be used simultaneously. Preferredcombinations include alum with MPL, alum with QS-21, MPL with QS-21, andalum, QS-21 and MPL together. Also, Incomplete Freund's adjuvant can beused (Chang et al., 1998), optionally in combination with any of inQS-2, and WPL and all combinations thereof.

The antigenic product of the present invention is often administered aspharmaceutical compositions comprising an active agent, i.e., theantigenic product, and a variety of other pharmaceutically acceptablecomponents. See Remington's Pharmaceutical Science (15^(th) ed., MackPublishing Company, Easton, Pa., 1980). The preferred form depends onthe intended mode of administration and therapeutic application. Thecompositions can also include, depending on the formulation desired,pharmaceutically acceptable, non-toxic carriers or diluents, which aredefined as vehicles commonly used to formulate pharmaceuticalcompositions for animal or human administration. The diluent is selectedso as not to affect the biological activity of the combination. Examplesof such diluents are distilled water, physiological phosphate-bufferedsaline, Ringer's solutions, dextrose solution, and Hank's solution. Inaddition, the pharmaceutical composition or formulation may also includeother carriers, adjuvants, or auxiliary agents or nontoxic,nontherapeutic, nonimmunogenic stabilizers and the like.

Pharmaceutical compositions can also include large, slowly metabolizedmacromolecules such as proteins, polysaccharides such as chitosan,polylactic acids, polyglycolic acids and copolymers (such as latexfunctionalized sepharose™, agarose, cellulose, and the like), polymericamino acids, amino acid copolymers, and lipid aggregates (such as oildroplets or liposomes). Additionally, these carriers can function asimmunostimulating agents (i.e., adjuvants).

For parenteral administration, the antigenic product of the presentinvention can be administered as injectable dosages of a solution orsuspension of the substance in a physiologically acceptable diluent witha pharmaceutical carrier that can be a sterile liquid such as wateroils, saline, glycerol, or ethanol. Additionally, auxiliary substances,such as wetting or emulsifying agents, surfactants, pH bufferingsubstances and the like can be present in compositions. Other componentsof pharmaceutical compositions are those of petroleum, animal,vegetable, or synthetic origin, for example, peanut oil, soybean oil,and mineral oil. In general, glycols such as propylene glycol orpolyethylene glycol are preferred liquid carriers, particularly forinjectable solutions.

Typically, compositions are prepared as injectables, either as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid vehicles prior to injection can also be prepared.The preparation also can be emulsified or encapsulated in liposomes ormicro particles such as polylactide, polyglycolide, or copolymer forenhanced adjuvant effect, as discussed above (see Langer, 1990 andHanes, 1997).

Patients amenable to treatment include individuals at risk ofAlzheimer's disease but not showing symptoms, as well as patientspresently showing symptoms. Virtually anyone is at risk of sufferingfrom Alzheimer's disease if he or she lives long enough. Therefore, thepresent antigenic product can be administered prophylactically to thegeneral population without the need for any assessment of the risk ofthe subject patient. The present methods are especially useful forindividuals who have a known genetic risk of Alzheimer's disease. Suchindividuals include those having relatives who have experienced thisdisease, and those whose risk is determined by analysis of genetic orbiochemical markers. Genetic markers of risk toward Alzheimer's diseaseinclude mutations in the APP gene, particularly mutations at position717 and positions 670 and 671 referred to as the Hardy and the Swedishfamilial AD mutations, respectively. Other markers of risk are mutationsin the presenilin genes, PS1 and PS2, and ApoE4, family history of AD,hypercholesterolemia or atherosclerosis. Individuals presently sufferingfrom Alzheimer's disease can be recognized from characteristic dementia,as well as the presence of risk factors described above. In addition, anumber of diagnostic tests are available for identifying individuals whohave AD. These include measurement of CSF tau and Aβ42 levels. Elevatedtau and decreased Aβ42 levels signify the presence of AD. Individualssuffering from Alzheimer's disease can also be diagnosed by ADRDAcriteria.

In asymptomatic patients, treatment can begin at any age (e.g., 10, 20,30). Usually, however, it is not necessary to begin treatment until apatient reaches 40, 50, 60 or 70. Treatment typically entails multipledosages over a period of time. Treatment can be monitored by assayingantibody, or B-cell responses to the antigenic product of the presentinvention over time. If the response falls, a booster dosage isindicated. In the case of potential Down's syndrome patients, treatmentcan begin antenatally by administering therapeutic agent to the motheror shortly after birth.

The present invention also provides for methods of detecting an immuneresponse against the β-secretase cleavage site of AβPP in a patientsuffering from or susceptible to Alzheimer's disease. The methods areparticularly useful for monitoring a course of treatment beingadministered to a patient. The methods can be used to monitor boththerapeutic treatment on symptomatic patients and prophylactic treatmenton asymptomatic patients by monitoring antibody produced in response toadministration of immunogen.

Some methods entail determining a baseline value of an immune responsein a patient before administering a dosage of the antigenic product, andcomparing this with a value for the immune response after treatment. Asignificant increase (i.e., greater than the typical margin ofexperimental error in repeat measurements of the same sample, expressedas one standard deviation from the mean of such measurements) in valueof the immune response signals a positive treatment outcome (i.e., thatadministration of the agent has achieved or augmented an immuneresponse). If the value for immune response does not changesignificantly, or decreases, a negative treatment outcome is indicated.In general, patients undergoing an initial course of treatment with animmunogenic agent are expected to show an increase in immune responsewith successive dosages, which eventually reaches a plateau.Administration of agent is generally continued while the immune responseis increasing. Attainment of the plateau is an indicator thatadministration of the immunogen can be discontinued or reduced in dosageor frequency.

In other methods, a control value (i.e., a mean and standard deviation)of immune response is determined for a control population. Typically theindividuals in the control population have not received prior treatment.Measured values of immune response in a patient after administering theantigenic product are then compared with the control value. Asignificant increase relative to the control value (e.g., greater thanone standard deviation from the mean) signals a positive treatmentoutcome. A lack of significant increase or a decrease signals a negativetreatment outcome. Administration of agent is generally continued whilethe immune response is increasing relative to the control value. Asbefore, attainment of a plateau relative to control values is anindicator that the administration of treatment can be discontinued orreduced in dosage or frequency.

In other methods, a control value of immune response (e.g., a mean andstandard deviation) is determined from a control population ofindividuals who have undergone treatment with the antigenic product andwhose immune responses have plateaued in response to treatment. Measuredvalues of immune response in a patient are compared with the controlvalue. If the measured level in a patient is not significantly different(e.g., more than one standard deviation) from the control value,treatment can be discontinued. If the level in a patient issignificantly below the control value, continued administration of agentis warranted. If the level in the patient persists below the controlvalue, then a change in treatment regime, for example, use of adifferent adjuvant may be indicated.

In other methods, a patient who is not presently receiving treatment buthas undergone a previous course of treatment is monitored for immuneresponse to determine whether a resumption of treatment is required. Themeasured value of immune response in the patient can be compared with avalue of immune response previously achieved in the patient after aprevious course of treatment. A significant decrease relative to theprevious measurement (i.e., greater than a typical margin of error inrepeat measurements of the same sample) is an indication that treatmentcan be resumed. Alternatively, the value measured in a patient can becompared with a control value (mean plus standard deviation) determinedin a population of patients after undergoing a course of treatment.Alternatively, the measured value in a patient can be compared with acontrol value in populations of prophylactically treated patients whoremain free of symptoms of disease, or populations of therapeuticallytreated patients who show amelioration of disease characteristics. Inall of these cases, a significant decrease relative to the control level(i.e., more than a standard deviation) is an indicator that treatmentshould be resumed in a patient.

The tissue sample for analysis is typically blood, plasma, serum, mucousor cerebrospinal fluid from the patient. The sample is analyzed forindication of an immune response to the antigenic product. The immuneresponse can be determined from the presence of antibodies thatspecifically bind to the β-secretase cleavage site of AβPP, i.e., ELISA.

A further aspect of the present invention provides for antibodies raisedagainst the AβPP epitope spanning the β-secretase cleavage site of AβPPas carried on the antigenic product in the immunizing compositionaccording to the present invention and for molecules that includes theantigen-binding portion of such antibodies.

It should be understood that when the term “antibodies” is used withrespect to the antibody embodiments of the present invention, this isintended to include intact antibodies, such as polyclonal antibodies ormonoclonal antibodies (mAbs), as well as proteolytic fragments thereofsuch as the Fab or F(ab′)2 fragments. Furthermore, the DNA encoding thevariable region of the antibody can be inserted into other antibodies toproduce chimeric antibodies (see, for example, U.S. Pat. No. 4,816,567)or into T-cell receptors to produce T-cells with the same broadspecificity (see Eshhar, et al, 1990 and Gross et al, 1989).Single-chain antibodies can also be produced and used. Single-chainantibodies can be single-chain composite polypeptides having antigenbinding capabilities and comprising a pair of amino acid sequenceshomologous or analogous to the variable regions of an immunoglobulinlight and heavy chain (linked VH-VL or single-chain FV). Both VH and VLmay copy natural monoclonal antibody sequences or one or both of thechains may comprise a CDR-FR construct of the type described in U.S.Pat. No. 5,091,513 (the entire content of which is hereby incorporatedherein by reference). The separate polypeptides analogous to thevariable regions of the light and heavy chains are held together by apolypeptide linker. Methods of production of such single-chainantibodies, particularly where the DNA encoding the polypeptidestructures of the VH and VL chains are known, may be accomplished inaccordance with the methods described, for example, in U.S. Pat. Nos.4,946,778, 5,091,513 and 5,096,815, the entire contents of each of whichare hereby incorporated herein by reference.

An antibody is said to be “capable of binding” a molecule if it iscapable of specifically reacting with the molecule to thereby bind themolecule to the antibody. The term “epitope” is meant to refer to thatportion of any molecule capable of being bound by an antibody which canalso be recognized by that antibody. Epitopes or “antigenicdeterminants” usually consist of chemically active surface groupings ofmolecules such as amino acids or sugar side chains and have specificthree dimensional structural characteristics as well as specific chargecharacteristics.

Polyclonal antibodies are heterogeneous populations of antibodymolecules derived from the sera of animals immunized with an antigen.

Monoclonal antibodies (mAbs) are a substantially homogeneous populationof antibodies to specific antigens. MAbs may be obtained by methodsknown to those skilled in the art. See, for example Kohler et al,(1975); U.S. Pat. No. 4,376,110; Harlow et al, (1988); and Colligan etal, (2001), the entire contents of which references are incorporatedentirely herein by reference. Such antibodies may be of anyimmunoglobulin class including IgG, IgM, IgE, IgA, and any subclassthereof. The hybridoma producing the mAbs of this invention may becultivated in vitro or in vivo. High titers of mAbs can be obtained byin vivo production where cells from the individual hybridomas areinjected intraperitoneally into pristane-primed Balb/c mice to produceascites fluid containing high concentrations of the desired mAbs. MAbsof isotype IgM or IgG may be purified from such ascites fluids, or fromculture supernatants, using column chromatography methods well known tothose of skill in the art.

Chimeric antibodies are molecules, the different portions of which arederived from different animal species, such as those having a variableregion derived from a murine mAb and a human immunoglobulin constantregion. Chimeric antibodies are primarily used to reduce immunogenicityduring application and to increase yields in production, for example,where murine mAbs have higher yields from hybridomas but higherimmunogenicity in humans, such that human/murine chimeric or humanizedmAbs are used. Chimeric and humanized antibodies and methods for theirproduction are well-known in the art, such as Cabilly et al (1984),Morrison et al (1984), Boulianne et al (1984), Cabilly et al, EuropeanPatent 0 125 023 (1984), Neuberger et al (1985), Taniguchi et al,European Patent 0 171 496 (1985), Morrison et al, European Patent 0 173494 (1986), Neuberger et al, WO 8601533 (1986), Kudo et al, EuropeanPatent 0 184 187 (1986), Sahagan et al (1986); Robinson et al, WO9702671 (1987), Liu et al (1987), Sun et al (1987), Better et al (1988),and Harlow et al (1988). These references are hereby incorporated hereinby reference.

A “molecule which includes the antigen-binding portion of an antibody,”is intended to include not only intact immunoglobulin molecules of anyisotype and generated by any animal cell line or microorganism, orgenerated in vitro, such as by phage display technology for constructingrecombinant antibodies, but also the antigen-binding reactive fractionthereof, including, but not limited to, the Fab fragment, the Fab′fragment, the F(ab′)₂ fragment, the variable portion of the heavy and/orlight chains thereof, and chimeric or single-chain antibodiesincorporating such reactive fraction, or molecules developed to delivertherapeutic moieties by means of a portion of the molecule containingsuch a reactive fraction. Such molecules may be provided by any knowntechnique, including, but not limited to, enzymatic cleavage, peptidesynthesis or recombinant techniques.

An increasing body of evidence shows that olfactory deficits anddegenerative changes in the central olfactory pathways are affectedearly in the clinical course of AD. Moreover, the anatomic patternsinvolved in AD suggest that the olfactory pathway may be the initialstage in the development of AD.

Olfactory receptor neurons are bipolar cells that reside in theepithelial lining of the nasal cavity. Their axons traverse thecribriform plate and project to the first synapse of the olfactorypathway in the olfactory bulb of the brain. The axons of olfactoryneurons from the nasal epithelium form bundles of 1000 amyelinic fibers.This configuration makes them a highway by which viruses or othertransported substances may gain access to the CNS across the BBB.

In the early stages of AD, the BBB may limit the entry of antibodycirculating in the periphery to the CNS. In contrast, single chainantibodies or molecules having an antigen-binding portion of an antibodydirected against an epitope spanning the β-secretase cleavage site ofAβPP, which antibodies or molecules are displayed on a phage surfacehave the potential not only be delivered directly to the CNS byintranasal administration but also to prevent olfactory permanent damageby Aβ in the patients. As previously shown, intranasal administration(Mathison et al., 1998; Chou et al., 1997 and Draghia et al., 1995)enables the direct entry of viruses and macromolecules into the CSF orCNS.

Use of olfactory receptor neurons as a point of delivery for anadenovirus vector to the brain is reported in the literature. Thismethod reportedly causes expression of a reporter gene in the brain for12 days without apparent toxicity (Draghia et al., 1995).

Thus, according to the method for passive immunization according to thepresent invention, a vehicle displaying an immunological antigen-bindingportion of an antibody capable of blocking the β-secretase cleavage siteof AβPP is delivered via this route to the brain.

As Aβ is produced continuously by cells in peripheral tissues whichcross the blood brain barrier (BBB) leading to localized toxic effectsin specific neuronal populations, intranasal administration of such avehicle may also prevent the progression of the disease by minimizingthe amount of peripheral Aβ available to form plaques.

Antibody phage or virus display is accomplished, for example, by fusingthe coding sequence of the antibody variable regions to a phage or viruscoat protein. To this end, the variable (V) regions (V_(H) and V_(L))mRNA isolated from antibody-producing cells is reverse-transcribed intocDNA, and heavy and light chains assembled randomly to encode singlechain Fv (scFv). These cassettes are cloned directly into a suitablevector such as a phagemid vector for expression and display on the phageor virus surface. This linkage between antibody genotype and phenotypeallows the enrichment of antigen specific phage or virus antibodies,using immobilized or labeled antigen. Phage or virus that display arelevant antibody will be retained on a surface coated with antigen,while non-adherent phages or viruses will be washed away. Bound phagesor viruses can be recovered from the surface, re-infected into suitablehost cells and re-grown for further enrichment and, eventually forbinding analysis.

The success of antibody phage or virus display hinges on the combinationof this display and enrichment method. Phage or virus antibody genes canbe sequenced, mutated and screened to improve antigen binding.

It is possible to rearrange the genes which code for the various regionsof an antibody molecule such that its specificity and affinity for anantigen are altered. The antibody can be maintained on the surface ofthe phage or virus for further manipulation or be released as solublescFv (˜25 kDa) fragment.

Since its invention at the beginning of the 1990's, antibody phagedisplay has revolutionized the generation of monoclonal antibodies andtheir engineering. This is because phage display allows antibodies to bemade completely in vitro, bypassing the immune system and theimmunization procedure, and allowing in vitro tailoring of the affinityand specificity of the antibody. It is therefore anticipated that themost efficient new vaccine development strategies will employ thistechnology.

Additional features can be added to the vector to ensure its safetyand/or enhance its therapeutic efficacy. Such features include, forexample, markers that can be used to negatively select against cellsinfected with the recombinant virus such as antibiotic sensitivity.Negative selection is therefore a means by which infection can becontrolled because it provides inducible suicide through the addition ofantibiotic. Such protection ensures that if, for example, mutationsarise that produce altered forms of the viral vector or recombinantsequence, cellular transformation will not occur. Features that limitexpression to particular cell types can also be included. Such featuresinclude, for example, promoter and regulatory elements that are specificfor the desired cell type.

Viruses are very specialized infectious agents that have evolved, inmany cases, to elude host defense mechanisms. Typically, viruses infectand propagate in specific cell types. The targeting specificity of viralvectors utilizes its natural specificity to specifically targetpredetermined cell types and thereby introduce a recombinant gene intothe infected cell.

The direct brain delivery of antibodies overcomes crossing the BBB byusing olfactory neurons as transporters to the brain. In the olfactoryepithelium, the dendrites of the primary olfactory neurons are incontact with the nasal lumen, and via the axons, these neurons are alsoconnected to the olfactory bulbs of the brain. Phages that come intocontact with the olfactory epithelium can be taken up in the primaryolfactory neurons and be transported to the olfactory bulbs, and evenfurther into other areas of the brain.

A further aspect of the present invention provides a pharmaceuticalcomposition containing a pharmaceutically acceptable carrier, excipient,diluent, or auxiliary agent and the viral display vehicle displaying onits surface a single chain antibody directed against an epitope spanningthe β-secretase cleavage site of AβPP.

Having now generally described the invention, the same will be morereadily understood through reference to the following example which isprovided by way of illustration and is not intended to be limiting ofthe present invention.

EXAMPLE

Materials and Results

Immunization:

3 groups of Balb/c mice were injected with 3 different MAP(octa-branched) conjugated peptides: ISEVKMDA (residues 1-8 of SEQ IDNO:1, where residue 6 is Met), VKMDAEFRH (SEQ ID NO:5) and ISEVKLDA(residues 1-8 of SEQ ID NO:1, where residue 6 is Leu) . The stocksolution (2 mg/ml) was prepared as follows: 1000 μl of double distilledwater (DDW), 665 μl of Freund's adjuvant (Complete Freund's adjuvant onfirst injection and Incomplete Freund's adjuvant on subsequentinjections) and 335 μl of peptide stock solution. 300 μl of thevaccination solution (immunizing composition) was injected into eachmouse every two weeks after initial injection. The highest immuneresponse raised against MAP-ISEVKLDA (residues 1-8 of SEQ ID NO:1, whereresidue 6 is Leu) is shown in FIG. 3.

ELISA Procedure for IgG Titer Quantification:

96-well ELISA plates were coated with 50 μl/well peptide stock solutionat dilution of 1:500 in coating buffer (0.1M Na₂CO₃, pH 9.6) andincubated overnight at 4° C. The plates were washed with 2×PBS (0.05%TWEEN) and 2×PBS, blocked with 3% milk/PBS 180 μl/well and incubated for1.5 hr at 37° C. Serum dilutions in 1% milk/PBS 50 μl/well wereincubated for 1 hr at 37° C., then washed again with 50 μl/wellα-mouse-IgG (H+L)HRP conjugated with dilution of 1:5000 in 1% milk/PBSincubated for 1 hr at 37° C. Additional washings contained PBS (0.05%TWEEN) and finally PBS. Reaction was done with 50 μl/well of 15 ml 0.05Mcitrate buffer with 30 mg OPD and 5 μl of 30% H₂O₂. Reaction time was5-10 minutes and was then stopped by addition of 25 μl/well 4 M HCl.

Cell Line:

Cell Culture-Chinese hamster ovary (CHO) cells were grown in Dulbecco'smodified Eagle's medium (F-12) containing 10% fetal calf serum (FCS) and2.5 mM L-glutamine. Stably transfected CHO cell lines expressing wildtype AβPP 751 were generated with expression vector pCMV751 usingLipofectin-mediated transfection (Life Technologies, Inc., Gaithersburg,Md.) and selected by G418 resistance. A 6-well plate was then seededwith 2.5×10⁶ to 4×10⁶ cells from each transfected cell line. Followingovernight incubation, serum-free medium was added to each well and thenthe cells were incubated in a solution of anti-β-secretase cleavage siteon AβPP serum and non-injected mouse serum as control, then incubatedfor 48 h. Media were collected from each well and subjected to ELISA.

Two Site-Sandwich Aβ ELISA:

A two site-sandwich ELISA was used to measure Aβ production andsecretion from the above serum-treated and untreated cells. Themonoclonal anti-Aβ antibody 266 was used as a capture antibody.Ninety-six well plates were coated with a solution of 266 (0.1 μg/well)and 0.1M carbonate buffer (0.1M Na₂CO₃, pH 9.6), and incubated overnightat 4° C. The plates were washed with 2×PBSt (0.05% TWEEN) and 2×PBS,subsequently blocked with 180 μl/wall 3% BSA/PBS and incubated for 2.5hr. at 37° C., then washed as above. Biotinylated monoclonal anti-Aβantibody 6C6 (125 ng/well) for total Aβ and biotinylated monoclonalanti-Aβ1-42 antibody 8G7 (25 ng/well) for Aβ1-42 specific, both in 1%BSA/PBS, were used for detection. Plates were washed andavidin-conjugated alkaline phosphatase (Sigma, St. Louis, Mo.) (1μg/well) was added for 2 hr at room temperature, then they were washedin 3×PBSt (0.05% TWEEN) and 4×PBS. The substrate p-nitrophenylphosphate(PnPP; Sigma) was used as the reporter system. Reaction was done with 50μl/well (15 ml) of diethanolamine buffer with 30 mg PnPP. PnPPfluorescence was examined at wavelength of 405 nm. For construction ofstandard curves the Aβ standard (1-28) and Aβ standard (1-42) wereprepared in the presence of protease inhibitors and 1% BSA in serum-freemedium or extraction buffer (FIG. 4). FIG. 4 shows the inhibition oftotal amyloid beta peptide (Aβ) secretion to growing media as measuredby ELISA.

Quantification of Intracellular Aβ (1-42)

CHO cells were collected from each well using cell scraper in theirgrowing media. The collected media were centrifuged at 3000 g for 2 min,collected cells were washed with PBS and centrifuged twice. Cells weresuspended in 100 μl 70% formic acid and sonicated for 10 sec with probesonicator. The solution was centrifuged at 100,000 g for 20 min at 4° C.to remove insoluble material; supernatant was neutralized with 1.9 ml 1MTRIS. Samples of this solution were diluted 1:3 in H₂O and 300 μl of itwas added for the ELISA, as described above. After only 5 daysincubation, considerable inhibition of Aβ (1-42) accumulation, asmeasured by ELISA, was found (FIG. 5).

Confocal Microscopy for Co-Localization of AβPP-Sera Antibody Complexwith BACE (β-Secretase) into the Cells:

CHO cells overexpressing AβPP 751 were grown for 24 hr in 6-wellsplates, washed with 3×PBS and fixed with 4% (in PBS) paraformaldehydefor 30 min at room temperature. Cells were washed as above andpermeabilized by adding 0.3% TRITON-100 in 1% BSA/PBS for 5 min, thenwashed with 0.5% 3× (BSA/PBS). Non-specific binding with rabbit serum1:150 for 2 hr was followed by washing as described above. Anti-βsecretase cleavage site on AβPP serum or α-BACE1 (raised against theβ-secretase enzyme itself and supplied by Calbiochem, San Diego,Calif.), in dilution of 1:2000, was added, followed by incubation for 2hr, washed as described above and subjected to secondary antibody, asfollows: α-mouse-Cy3 for anti-β secretase site on AβPP serum and/orα-rabbit-FITC for α-BACE1. In FIG. 6, confocal microscopy of anti-βsecretase cleavage site on AβPP antibodies and BACE antibodies showedco-localization (light spots) in the cell perinuclear region.

Immunfluorescence Microscopy for Internalization Assay of AntibodiesAgainst β-Secretase Cleavage Site:

CHO cells overexpressing AβPP 751 were grown for 24 hr in 6-well plates.After washing, new media containing anti-β secretase cleavage site onAβPP serum in dilution of 1:500 were added. Cells were incubated for 30min and then washed with 3×PBS and fixed with 4% (in PBS)paraformaldehyde for 30 min at room temperature. Cells were washed asabove and divided into permeabilized cells by adding 0.3% TRITON-100 in1% BSA/PBS and incubated for 5 min. As control, untreated cells werewashed with 0.5% 3× (BSA/PBS). The blocking step was done with 3% BSAfor 2 hr followed by washing step as above. Secondary antibody wasincubated for 1 hr at room temperature in the dark, after which it waswashed with 3×PBS and mounted with ProLong anti-fade kit (MolecularProbes, Eugene, Oreg.). FIG. 7A shows the immunostaining of internalizedanti-β-secretase cleavage site AβPP antibody after fixation andpermeabilization. FIG. 7B is the control.

Inhibition of Plaque Formation in AβPP Transgenic Mice:

6 mice were immunized as describe above and 3 mice were used as control.After five months of immunization, the mice were sacrificed and brainslices were subjected for standard ThS protocol plaque staining. Plaquenumber was counted under microscope examination and a reduction ofplaque number in transgenic mice immunized with the antigen, compared tountreated mice, was observed (FIG. 8).

Having now fully described this invention, it will be appreciated bythose skilled in the art that the same can be performed within a widerange of equivalent parameters, concentrations, and conditions withoutdeparting from the spirit and scope of the invention and without undueexperimentation.

While this invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications. This application is intended to cover any variations,uses, or adaptations of the inventions following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth as follows in the scope of theappended claims.

All references cited herein, including journal articles or abstracts,published or corresponding U.S. or foreign patent applications, issuedU.S. or foreign patents, or any other references, are entirelyincorporated by reference herein, including all data, tables, figures,and text presented in the cited references. Additionally, the entirecontents of the references cited within the references cited herein arealso entirely incorporated by references.

Reference to known method steps, conventional methods steps, knownmethods or conventional methods is not in any way an admission that anyaspect, description or embodiment of the present invention is disclosed,taught or suggested in the relevant art.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingknowledge within the skill of the art (including the contents of thereferences cited herein), readily modify and/or adapt for variousapplications such specific embodiments, without undue experimentation,without departing from the general concept of the present invention.Therefore, such adaptations and modifications are intended to be withinthe meaning and range of equivalents of the disclosed embodiments, basedon the teaching and guidance presented herein. It is to be understoodthat the phraseology or terminology herein is for the purpose ofdescription and not of limitation, such that the terminology orphraseology of the present specification is to be interpreted by theskilled artisan in light of the teachings and guidance presented herein,in combination with the knowledge of one of ordinary skill in the art.

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1. An immunizing composition, comprising an immunizing effective amountof an antigenic product which induces an immune response against anepitope that spans the β-secretase cleavage site of amyloid precursorprotein (AβPP) so as to inhibit cleavage of AβPP by β-secretase, and apharmaceutically acceptable carrier, diluent, excipient, adjuvant, orauxiliary agent.
 2. The immunizing composition of claim 1, wherein saidantigenic product is encapsulated in a liposome.
 3. A method forinducing an immune response against the β-secretase cleavage site ofAβPP, comprising administering the immunizing composition of claim 1 toa subject to induce an immune response against the β-secretase cleavagesite of AβPP and inhibit β-secretase cleavage of AβPP.
 4. The immunizingcomposition of claim 1, wherein said antigenic product comprises anantigenic peptide comprising an AβPP epitope that spans the β-secretasecleavage site of AβPP.
 5. The immunizing composition of claim 4, whereinsaid antigenic peptide consists of residues 1 to 8 of SEQ ID NO:1. 6.The immunizing composition of claim 4, wherein said antigenic peptideconsists of the amino acid sequence of SEQ ID NO:5.
 7. The immunizingcomposition of claim 4, wherein said antigenic peptide comprisesresidues 1 to 8 of SEQ ID NO:1.
 8. The immunizing composition of claim7, wherein the residue at position 6 of SEQ ID NO:1 is Met.
 9. Theimmunizing composition of claim 7, wherein the residue at position 6 ofSEQ ID NO:1 is Leu.
 10. The immunizing composition of claim 4, whereinsaid antigenic peptide comprises the amino acid sequence of SEQ ID NO:5.11. The immunizing composition of claim 4, wherein said antigenicpeptide comprises two overlapping AβPP epitopes that both span theβ-secretase cleavage site of AβPP.
 12. The immunizing composition ofclaim 11, wherein said two overlapping AβPP epitopes are identical. 13.The immunizing composition of claim 1, wherein said antigenic productcomprises a display vehicle and an antigenic peptide displayed on saiddisplay vehicle, said antigenic peptide comprising an AβPP epitope thatspans the β-secretase cleavage site of AβPP.
 14. The immunizingcomposition of claim 13, wherein said display vehicle comprises adendritic polymer, built on a core molecule, which is at leastdifunctional so as to provide branching, and containing up to 16terminal functional groups to which said antigenic peptide is joined bycovalent bonds.
 15. The immunizing composition of claim 14, wherein saiddendritic polymer contains eight terminal functional groups to which anantigenic peptide is joined.
 16. The immunizing composition of claim 14,wherein said antigenic peptide comprises residues 1 to 8 of SEQ ID NO:1.17. The immunizing composition of claim 16, wherein the residue atposition 6 of SEQ ID NO:1 is Met.
 18. The immunizing composition ofclaim 16, wherein the residue at position 6 of SEQ ID NO:1 is Leu. 19.The immunizing composition of claim 14, wherein said antigenic peptidecomprises the amino acid sequence of SEQ ID NO:5.
 20. The immunizingcomposition of claim 14, wherein said antigenic peptide comprises twooverlapping AβPP epitopes that both span the β-secretase cleavage siteof AβPP.
 21. The immunizing composition of claim 20, wherein said twooverlapping AβPP epitopes are identical.
 22. The immunizing compositionof claim 14, wherein said core molecule is lysine.
 23. The immunizingcomposition of claim 14, further comprising a molecule having adjuvantproperties joined to said dendritic polymer.
 24. The immunizingcomposition of claim 13, wherein said display vehicle comprises a viraldisplay vehicle displaying on its surface said antigenic peptide. 25.The immunizing composition of claim 24, wherein said viral displayvehicle is a filamentous bacteriophage.
 26. The immunizing compositionof claim 24, wherein said antigenic peptide comprises residues 1 to 8 ofSEQ ID NO:1.
 27. The immunizing composition of claim 24, wherein saidantigenic peptide comprises the amino acid sequence of SEQ ID NO:5. 28.An isolated antigenic peptide consisting of 6-14 amino acid residues ofthe amyloid precursor protein (AβPP) that span the β-secretase cleavagesite of AβPP.
 29. The isolated antigenic peptide of claim 28, whereinsaid antigenic peptide comprises residues 1 to 8 of SEQ ID NO:1.
 30. Theisolated antigenic peptide of claim 29, wherein the residue at position6 of SEQ ID NO:1 is Met.
 31. The isolated antigenic peptide of claim 29,wherein the residue at position 6 of SEQ ID NO:1 is Leu.
 32. Theisolated antigenic peptide of claim 28, wherein said antigenic peptideconsists of 1 to 8 of SEQ ID NO:1.
 33. The isolated antigenic peptide ofclaim 28, wherein said antigenic peptide comprises the amino acidsequence of SEQ ID NO:5.
 34. The isolated antigenic peptide of claim 28,wherein said antigenic peptide consists of the amino acid sequence ofSEQ ID NO:5.